Some embodiments described herein relate to devices and methods for sample characterization and various uses thereof.
Separation of analyte components from a more complex analyte mixture on the basis of an inherent quality of the analytes, and providing sets of fractions that are enriched for states of that quality is a key part of analytical chemistry. Simplifying complex mixtures in this manner reduces the complexity of downstream analysis. It can be advantageous to perform two or more enrichment steps that are orthogonal, (e.g., based on different and/or unrelated qualities). In many cases, however, the process of performing orthogonal enrichment steps using known methods and/or devices is cumbersome, and can dilute the analyte beyond the sensitivity of the downstream analytical equipment. In addition, complications can arise when attempting to interface known enrichment methods and/or devices with analytical equipment and/or techniques.
Methods have been used to interface protein sample preparation techniques with downstream detection systems such as mass spectrometers. A common method is to prepare samples using liquid chromatography and collect fractions for mass spectrometry (LC-MS). This has the disadvantage of requiring protein samples to be digested into peptide fragments, leading to large number of sample fractions which must be analyzed and complex data reconstruction post-run. While certain forms of liquid chromatography can be coupled to a mass spectrometer, for example peptide map reversed-phase chromatography, these known techniques are restricted to using peptide fragments, rather than intact proteins, which limit their utility.
Another method to introduce samples into a mass spectrometer is electrospray ionization (ESI). In ESI, small droplets of sample and solution at a distal end of a capillary or microfluidic device are ionized to induce an attraction to the charged plate of a mass spectrometer. The droplet then stretches in this induced electric field to a cone shape (“Taylor cone”), which then releases small droplets into the mass spectrometer for analysis. Typically, this is done in a capillary, which provides a convenient volume and size for ESI. Capillaries however, provide a linear flow path that does not allow for multi-step processing.
Other work has been pursued with microfluidic devices. Microfluidic devices may be produced by various known techniques and provide fluidic channels of defined width that can make up a channel network designed to perform different fluid manipulations. These devices offer an additional level of control and complexity than capillaries. In connection with ESI, known devices include outwardly tapered tips and conductive edges in an attempt to enhance the ESI in these devices. The outward taper of known microfluidic devices used for ESI, however, exposes the fragile Taylor cone structure to potential disturbances by turbulent air flow and results in a contact surface geometry that will support only a limited range of cone radii, which limits control over the volume introduced to the mass spectrometer through ESI. Additionally, electrolysis of water at the conductive edge can lead to gas bubble formation, which interferes with the cone development.
One application for protein mass spectrometry is for characterization during the development and manufacturing of biologic and biosimilar pharmaceuticals. Biologics and biosimilars are a class of drugs which include, for example, recombinant proteins, antibodies, live virus vaccines, human plasma-derived proteins, cell-based medicines, naturally-sourced proteins, antibody-drug conjugates, protein-drug conjugates and other protein drugs.
Regulatory compliance demands that biologics require extensive testing during development and manufacture that is not required for small molecule drugs. This is because the manufacture of biologics has greater complexity due to, for example, using living material to produce the biologic, greater complexity of biologic molecule, greater complexity of the manufacturing process. Characteristics required to be defined include, for example, charge, efficacy, hydrophobic changes, mass, and glycosylation. Currently these tests are done independent of each other leading to a very time consuming and expensive process of characterizing biologics.